Fig. 5

C3a-peptide combined with C3aR promotes F-actin aggregation and spindle dynamics by directly binding to MYO10. A Triple immunofluorescence staining shows that C3a-peptide treatment increased C3aR expression (green) and enhanced F-actin aggregation in the cytoplasm, sub-cortical regions, and around the spindle (pink). C3aR and tubulin co-localized to the spindle (three independent biological replicates). B F-actin probes in living cells revealed that C3a-peptide treatment enhanced F-actin aggregation in the sub-cortical regions and around the spindle. C W/D, L/D, S-C, S1, S2, and cortical thickness were calculated and compared between the untreated and C3a-peptide-treated groups. D Triple immunofluorescence staining showed that the enhanced F-actin positive signals in the cytoplasm, sub-cortical regions, and around the spindle (pink) caused by C3a-peptide treatment were restored with a C3aR morpholino injection (three independent biological replicates). E Direct interaction between C3aR and MYO10 in mouse oocytes was determined with a proximity ligation assay (PLA) using rabbit anti-C3aR and anti-MYO10 antibodies. After staining, the oocytes were imaged by confocal and differential interference contrast (DIC) microscopy. Scale bar, 20 μm. F W/D, L/D, S-C, S1, S2, and cortical thickness were calculated and compared between the C3a-peptide-treated group and the combined C3a-peptide-treated and C3aR morpholino-injected group. G Confirming the direct interaction between C3aR and MYO10 by Co-IP (three independent biological replicates). Data are presented as the means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001 as compared to control cells